Author/Editor		Frol, Senta; Hren, Martin
Title		Izbira primernih monoklonskih protiteles in optimizacija imunohistokemijske metode za dokazovanje patološkega prionskega proteina v možganih bolnikov s sporadično obliko Creutzfeldt-Jakobove bolezni
Translated title		Selection of suitable monoclonal antibodies and optimization of the immunohistochemical method for the detection of pathological prion protein in the brains patients with sporadic Creutzfeldt-Jakob disease
Type		članek
Source		Med Razgl
Vol. and No.		Letnik 41, št. 1
Publication year		2002
Volume		str. 3-12
Language		slo
Abstract		Reliable diagnosis of prion diseases is based on a positive immunohistachemical reaction (IHR) using antibodies against prion protein (PrP). Unfortunately, this does not distinguish between cellular PrP (PrPc) and the pathological isoform (PrPsc). Consequently, this method requires intensive pre-treatments to remove PrPc and retrieve the epitope of PrPsc in paraffin sections of the brains of patients with prion disease. The aim of this study was to test anti-PrP monoclonal antibodies (MAb) V5B2, K4B3, A4/5 and E5/9, which were prepared at the Blood Transfusion Center of Slovenia, in order to compare them with commercially available MAb 3F4 and 6H4, and to design a simple antigen retrieval method to label PrPsc in immunohistochemistry. Sections of paraffin-embedded brain tissue for four cases of sporadic Creutzfeldt-Jakob disease (sCJD) and of two deceased patients of similar age who had not had CJD were treated lwith a modified standard ABC immunohistochemical method using our MAb, control MAb and four pre-treatments. The IHR of our MAb was compared with the IHR of the control MAb (3F4 and 6H4) with regard to the intensity of IHR and the number of positive reactions. The same MAb were tested on the frozen sections of two brains not affected by CJD without any pre-treatment. The results showed that all four tested MAbs were specific for PrPsc since, unlike the two control MAbs, they did not react with PrPc in the frozen sections of non-CJD brain. An improvement in IHR at more intense section pre-treatments of tissues was also demonstrated in terms of intensity and the number of positive reactions (p < 0.05). Our MAb V5B2 is more sensitive than the control MAb 3F4 (p=0.03). It does not need an equally intense pre-treatment as MAb 3F4 to label PrPsc in CJD-affected brain. (Abstract truncated at 2000 characters)
Summary		Diagnostika prionskih bolezni temelji na imunohistokemijskem določanju ob uporabi protiteles proti prionskemu proteinu (PrP), ki ne razlikujejo med celično obliko PrP (PrPc) in patološko izoobliko (PrPsc). Zato zahteva ta metoda močne predobdelave za odstranjevanje PrPc in razkrivanje epitopa na PrPsc v parafinskih rezinah umrlih za prionsko boleznijo. Namen naše naloge je bil testirati monoklonska protitelesa proti PrPsc (MPt) V5B2, K4B3, A4/5 in E5/9, ki so jih pripravili na Zavodu RS za transfuzijo krvi, jih primerjati s kontrolnimi, komercialno dostopnimi MPt 3F4 in 6H4, ter poiskati enostavnejšo predobdelavo za razkrivanje epitopa na PrPsc. Parafinske histološke rezine vzorcev možgan štirih umrlih za sporadično Creutzfeldt-Jakobovo boleznijo (sCJB) in dveh umrlih primerljive starosti, ki nista imela CJB, smo obdelali s prilagojeno standardno imunohistokemijsko metodo avidin-biotin kompleks (ABC) ob uporabi naših MPt, kontrolnih MPt in štirih predobdelav. Imunohistokemijsko reakcijo (IHR) z našimi MPt in PrPsc smo primerjali z IHR med kontrolnima MPt (3F4 in 6H4) in PrPsc z ozirom na jakost signala IHR in število pozitivnih reakcij. Ista MPt smo uporabili tudi na zaledenelih rezinah vzorcev dveh ne-CJB-možgan brez kakršnekoli predobdelave. V nalogi smo pokazali, da so vsa štiri testirana MPt specifična za Prsc, saj za razliko od dveh kontrolnih MPt niso prikazala PrPc v zaledenelih rezinah svežih ne-CJB-možgan. Prav tako smo pokazali, da se z močnejšimi predobdelavami histoloških rezin IHR izboljša, tako v jakosti kot tudi v številu pozitivnih reakcij (p < 0,05). Naše MPt V5B2 je občutljivejše od kontrolnega MPt 3F4 (p =0,03) in je zmožno z manj intenzivno predobdelavo zanesljivo označiti PrPsc v možganih obolelih za CJB. (Izvleček skrajšan pri 2000 znakih)
Descriptors		CREUTZFELDT-JAKOB SYNDROME PRIONS ANTIBODIES, MONOCLONAL IMMUNOHISTOCHEMISTRY